To define the molecular pathway of CTL cytotoxicity, we have tested the possibility that precursors of ICE-family proteases (caspases) are the physiologically relevant target granzyme substrates. Other labs have shown biochemically that granzyme B activates caspases which cause nuclear damage. We thus tested two types of caspase inhibitor for their ability to block both the granule exocytosis pathway and the Fas pathway of CTL cytotoxicity. We find that while cell death via the Fas ligand/Fas pathway and apoptotic nuclear damage induced by the granule exocytosis pathway requires caspases, target lysis via the granule exocytosis pathway does not. The first class of caspase inhibitor used was peptide fluoromethyl ketones-- ZVAD- FMK (an ICE inhibitor), BD-FMK (a CPP32 but not ICE inhibitor), and ZFA-FMK (a control cathepsin B inhibitor). None of these inhibits the asp-selective serine protease granzyme B. Both ZVAD-FMK and BD-FMK completely blocked target cell lysis (51Cr release) and apoptotic nuclear morphology induced by a Fas ligand bearing CTL hybridoma while ZFA-FMK did not, confirming a role for caspases in this death pathway. When the granule exocytosis pathway was examined using non-Fas bearing targets and conventional cloned CTL, these asp-containing FMK inhibitors completely blocked apoptotic nuclear morphology but had no significant effect on target lysis.These results have been confirmed with second type of caspase inhibitor, the Baculovirus protein p35, which blocks all known members of this family but not granzyme B. In these experiments we expressed p35 in target cells by transient transfection and measured the ability of CTL to release cotransfected-galactosidase. Although p35 effectively blocked lysis via the Fas pathway, lysis via the granule exocytosis pathway was not significantly inhibited. Thus we conclude that while caspases are part of the pathway inducing apoptotic nuclear damage for both CTL effector pathways, the granule exocytosis pathway uses another, more rapid, cell death pathway which is independent of caspases.